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Two years ago, genes coding for voltage-gated proton channels in humans, mice and Ciona intestinalis were discovered. Transfection of cDNA encoding the human HVCN1 (H(V)1) or mouse (mVSOP) ortholog of HVCN1 into mammalian cells results in currents that are extremely similar to native proton currents, with a subtle, but functionally important, difference. Expressed proton channels exhibit high H(+) selectivity, voltage-dependent gating, strong temperature sensitivity, inhibition by Zn(2+), and gating kinetics similar to native proton currents. Like native channels, expressed proton channels are regulated by pH, with the proton conductance-voltage (g(H)-V) relationship shifting toward more negative voltages when pH(o) is increased or pH(i) is decreased. However, in every (unstimulated) cell studied to date, endogenous proton channels open only positive to the Nernst potential for protons, E(H). Consequently, only outward H(+) currents exist in the steady state. In contrast, when the human or mouse proton channel genes are expressed in HEK-293 or COS-7 cells, sustained inward H(+) currents can be elicited, especially with an inward proton gradient (pH(o)pH(i)). Inward current is the result of a negative shift in the absolute voltage dependence of gating. The voltage dependence at any given pH(o) and pH(i) is shifted by about -30 mV compared with native H(+) channels. Expressed H(V)1 voltage dependence was insensitive to interventions that promote phosphorylation or dephosphorylation of native phagocyte proton channels, suggesting distinct regulation of expressed channels. Finally, we present additional evidence that speaks against a number of possible mechanisms for the anomalous voltage dependence of expressed H(+) channels.